EtBr and "clean" DNA

On Fri, 16 Jun 1995, Ole Skovgaard wrote:
> In article <9506121350.AA18506 at intnet.upj.com> pskaytes at INTNET.UPJ.COM writes:
> >From: pskaytes at INTNET.UPJ.COM> >Subject: Re: EtBr and "clean" DNA
> >Date: 12 Jun 1995 07:03:48 -0700
>> > The title is
> >"A 20-Minute Ethidium Bromide/High Salt Extraction Protocol for Plasmid
> >DNA", by Willem P.C. Stemmer, in Biotechniques, about 5 years ago.
>> >Good luck,
>> >Paul
>>> If anybody find the precise ref. please post it,
>> thanks
>> ole
>> ******************************************
> **** Ole Skovgaard ****
> **** Dept. Life. Sc. & Chemistry ****
> **** Roskilde University, DK ****
> ******************************************
>
This is a very handy wya of cleaning up DNA and works extremely well. I
do not have any reference with me, but here is what I do, for those who
are interested or are having trouble with sequencing, pcr, digests etc.:
1. DNA in 200 to 250uL.
2. Amm. acetate to 3.5M final conc. I use equal
vol of 7.M amm. acetate.
3. Add EtBr to final conc. 0.35mg/mL (approx.)
4. Mix by inverting or very slow and gentle vortexting. Let stand at RT
for 5 min.
5. Add equal vol of PCI and extract twice. This step removes about 80% of
the EtBr.
6. Extract with equal vol. of CI. The remainder of the EtBr should go by
now. If not, an extraction with TE-saturated butanol may be in order.
7. Ppt. the DNA with 2 vol. of EtOH and proceed from there as with any
other sample.
OBVIUOS NOTE: The concentration of the EtBr renders the DNA very
susceptible to UV damage, hence it is best to keep the tube wrapped in
foil or to work in a dimly lit area away from the window and/or the overhead
lab lightings.
>>>>>>>>>>>>>>>>>>>>>>>>>>>
Hiranya S. Roychowdhury
Plant Genetic Engineering Lab.
Box 3GL, NM State Univ.
Las Cruces, NM 88003
Phone: (505) 646-5785
hroychow at nmsu.edu
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